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Mycobacterium tuberculosis fusion protein and uses thereof

a technology of fusion protein and mycobacterium tuberculosis, which is applied in the field of genetically engineered mycobacterium tuberculosis fusion protein, can solve the problems of unconfirmed preventive effect of bcg, loss of original cultivar strain, and inability to protect those who have already been cultivated, so as to prevent the onset of tb, prolong the effect, and improve the immunity of the patien

Inactive Publication Date: 2012-05-08
YANGZHOU YILIN BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention is about a new M. tuberculosis fusion protein that can be used as a vaccine or an immunotherapy for tuberculosis. The fusion protein has the immunogenic activity of M. tuberculosis antigen and can be produced by a specific method involving a polynucleotide sequence and a host cell. The invention also includes a nucleic acid sequence coding for the fusion protein and a kit for diagnosing and treating tuberculosis. The technical effect of the invention is to provide a more effective and efficient tool for preventing and treating tuberculosis."

Problems solved by technology

With other reasons like wars, the originally cultivated strain may have already been lost.
However, the preventive effect of BCG has not been confirmed (Baily G V, et al, Ind. J. Med. Res., 72:1-74, 1980).
Moreover, BCG is useless in protecting those who have already been exposed to Mycobacteria in the environment, or who have already been infected by M. tuberculosis from TB (Brandt L., et al.
The high risk population always uses oral Isoniazid for prevention; however, it is usually hard to stick to as patients need to take it for a long time.
Moreover, Isoniazid often causes adverse reactions like peripheral neuropathy, and liver damage, as well as special adverse reactions, such as epilepsy, psychonosema, autonomis nervous disorders, etc (Guo L, et al.
Since attenuated bacteria strains have the potential of mutating back to its origins (Sampson S L, et al, Infect. Immun., 72:3031-3037, 2004), it is risky to vaccinate those who have compromised immune systems with live attenuated bacteria vaccines.
Live attenuated bacteria vaccines may also resemble BCG's fate, namely useless in protecting the infected population.
The use of a single kind of antigen does not give ideal results on protecting animals, which are infected by M. tuberculosis, from TB, while multiple single proteins or fusion proteins work better than a single kind of antigen.
In the past 40 years, there has been no novel TB chemotherapeutic drug on the market; and chemotherapies are facing the problem of consistently high rate of drug resistance.

Method used

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  • <i>Mycobacterium tuberculosis </i>fusion protein and uses thereof
  • <i>Mycobacterium tuberculosis </i>fusion protein and uses thereof
  • <i>Mycobacterium tuberculosis </i>fusion protein and uses thereof

Examples

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embodiments

[0085]In the following passage, referred embodiments in the present invention will be described in detail. However, these embodiments do not restrict the present invention.

example 1

The Preparation of M. tuberculosis Fusion Proteins

[0086]Two pairs of PCR primers were designed based on the Pab sequence of gene 38 KDa(Rv0934) and the gene sequence of ESAT-6(Rv3875). The primer upstream to Pab was 38 bases long, as shown in SEQ ID NO: 8. The protection bases and the NCOI restriction site (including the start codon ATG) were included in its 5′ end design. The primer downstream to Pab was 40 bases long, as shown in SEQ ID NO: 9. The BamHI restriction site and linker were included in its 5′ end design. The PCR amplification product of said primers was run through 1.2% agarose gel electrophoresis and the result showed a distinctively amplified band under ultraviolet light. Said band was around 1151 bp, which met the requirements of the design. The primer upstream to ESAT-6 was 32 bases long, as shown in SEQ ID NO: 10. The protection bases and BamHI restriction site were included in its 5′ end design. The primer downstream to ESAT-6 was 33 bases long, as shown in SEQ I...

example 2

Purification, Renaturation and Identification of the M. tuberculosis Fusion Protein of the Present Invention

[0089]First of all, lysis buffer was added into strains (3 ml of lysis buffer per 1 g of bacteria) to suspend them. Then, ultrasonic treatment with power of 200 W was performed to lyse bacteria. Each interval of ultrasonic treatments was 20 seconds, and 80 times of said treatments were separated with every treatment 20 seconds. Resulting solution from the previous step was then centrifuged under 4° C., 12 000 rpm for 15 minutes, the supernatant was discarded, and the inclusion bodies in the precipitation were washed once with lysis buffers 1% Triton-X100 (product of Sigma, bought from Beijing Jing Ke Hong Da Biotechnology Co., Ltd) and 2% Triton-X100 respectively. 50 mmol / L Tris.Cl / 8M urea (pH8.5) was then used to dissolve inclusion bodies under 4° C. until most inclusion bodies were dissolved. The resulted solution was centrifuged under 4° C., and 12 000 rpm for 15 minutes, a...

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Abstract

The present invention is related to a M. tuberculosis fusion protein, polynucleotide coding for said protein, and a vector and host cell that contain said polynucleotide. The present invention also involves the preparation of said fusion protein, and the use thereof in preventions and treatment of tuberculosis.

Description

FIELD OF THE INVENTION[0001]The present invention relates to a genetically engineered Mycobacterium tuberculosis fusion protein, and an application of said protein in developing new vaccines for tuberculosis (TB) preventions or immuno-treatments.BACKGROUND OF THE INVENTION[0002]Mycobacterium tuberculosis (M. tuberculosis) causes tuberculosis (TB). Each year, about 3 million people around the world are suffering from M. tuberculosis induced TB. China is one of the 22 countries around the world that have relatively high level of TB infections. In fact, following India, China has the second largest population of TB patients in the world. Recently, the number of people infected by TB in China is increasing again. According to the 4th nationwide TB epidemiological survey in 2000, China was rated high in several aspects: including the number of people infected (there are up to 550 million people infected, with the infection rate of 45%, which is significantly higher than the average infec...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C07K1/00
CPCA61K39/04C07K14/35A61K39/00A61K2039/55594C07K2319/40A61P31/06A61P37/04
Inventor HE, XIUYUNZHUANG, YUHUIWANG, HAIBINTAO, JIANHUA
Owner YANGZHOU YILIN BIOTECHNOLOGY CO LTD